In the fields related to life sciences such as genetics, immunology, molecular biology, and environmental science, flow cytometry is widely used to analyze microparticulate samples such as living cells, yeast, and bacteria. Particles or cells from 500 nm up to 50 micron can generally be measured in flow cytometry. In general, in the case of analyzing a cell or the like with a flow cytometer, a label made of a fluorescent substance is attached to the surface of a cell to be analyzed. Next, a liquid such as water or saline is used to move the labeled cell through a flow channel of a flow chamber, which is an area in which the labeled cell is to be analyzed, and laser light having a relatively high output is radiated towards a predetermined position to irradiate the cell. Then, forward-scattered light and side-scattered light, which are generated due to the size and structure of each cell, and fluorescence, which is generated by excitation light irradiation, are observed. In the case of observing fluorescence from a cell, a configuration for spectral analysis of the fluorescence condensed in a direction other than an irradiation path of excitation light is widely used to avoid adverse effects of transmitted or scattered excitation light. Fluorescent substances to be attached or combined for each type of cells are known. Accordingly, the wavelength and intensity of the fluorescence are observed and the intensity component to be superimposed is compensated to thereby identify the type of each cell flowing through the flow channel.
It is necessary to individually perform measurement of cells or the like with laser light. A large number of microparticulate samples are supplied to a flow chamber through a tube from a container such as a vial containing the samples. The flow chamber is generally configured to permit microparticulate samples to be aligned and flow by a method called hydrodynamic focusing.
Hydrodynamic focusing will be briefly described. When using hydrodynamic focusing, a sample flow including microparticulate samples is discharged from an elongated nozzle. The discharged sample flow is surrounded by a sheath flow of, e.g., water or saline, which is an isosmotic fluid, and flows through the flow channel of the flow chamber. The discharge pressure of the sample flow is set to be higher than that of the sheath flow, thereby permitting the microparticulate samples, which are randomly distributed, to be aligned and flow in the sample flow. This phenomenon is called a three-dimensional (3-D) laminar flow in terms of fluid dynamics. This makes it possible to radiate laser light independently towards each microparticulate sample, such as a cell, and to detect and analyze the scattered light and excited fluorescence.
Next, a typical flow cytometry system (“flow cytometer”) will be described. A typical flow cytometer includes a laser light irradiation optical system, a flow chamber, a detection optical system, and a control unit. The laser light irradiation optical system radiates laser light onto microparticulate samples within the flow chamber. The laser light irradiation optical system includes one or more lasers that output laser light having a wavelength corresponding to a label to be excited, and a condensing optical system that condenses the laser light on the flow chamber. The detection optical system can detect an intensity of light such as transmitted light, scattered light, and fluorescence from the microparticulate samples.
For example, consideration is given to the case where laser light is radiated in the vicinity of an orifice in a so-called jet-in-air system in which a stream is discharged through the orifice of a flow chamber. In this case, when laser light is radiated onto a stream having a cylindrical sectional shape from a direction substantially perpendicular to the section, the stream acts as a cylindrical lens. Accordingly, the laser light is radiated onto the microparticulate samples as a flat, elliptic beam in a direction perpendicular to the stream. An irradiation spot of laser light can have a substantially elliptical shape of 10 μm (minor axis)×70 μm (major axis), or other shapes or sizes. The irradiation spot is, e.g., an area of a microparticulate sample onto which enough laser light falls that characteristics of the microparticulate sample can be determined. Also in the case of using a flow chamber, which is called a cuvette, a flat beam having substantially the same width as that of a stream is used to observe particulate objects, e.g., microparticulate samples. Note that in the case of radiating a plurality of laser beams having different wavelengths, irradiation spots are generally disposed separately from each other in a flow direction for each wavelength so as to reduce stray light between the laser beams having different wavelengths.
When the flowing microparticulate samples pass through laser irradiation spots, scattered light and fluorescence, which is caused due to excitation of a labeled substance, are generated. The scattered light includes forward-scattered light having a small scattering angle which represents a size of a fine particle, and side-scattered light having a large scattering angle which represents an internal structure of a fine particle. Each of the forward-scattered light, the side-scattered light, and the fluorescence is detected by a photodetector of the detection optical system. The fluorescence has a small intensity and is radiated uniformly over the whole solid angle. For this reason, the fluorescence is condensed by a condenser lens having a large numerical aperture, and is then detected by an ultrasensitive photodetector which is called a photomultiplier tube (PMT). Then, the control unit performs amplification, analog-digital conversion, and operation on the light signal detected by the photodetector.
Furthermore, the above-described flow cytometer is provided with a mechanism for fractional extraction (sorting) of microparticulate samples such as cells. As a typical method, an ultrasonic vibration is applied to a stream in the flow chamber to thereby divide the stream, which is discharged from the orifice, into droplets, so that each droplet contains the microparticulate sample. Then, based on the measurement by the control unit, positive or negative electric charges are applied to the droplets. The droplets having positive or negative electric charges are deflected in the opposite direction depending on the polarity of the electric charges, when passing through a high-intensity electric field. After that, the deflected droplets are collected. As a result, sorted cells can be extracted for each type, and only cells of a specific type necessary for analysis, culture, or the like can be obtained. A flow cytometer having such a fractional extraction function is called a sorter. A flow cytometer which does not have such a fractional extraction function but has only an analysis function is called an analyzer.
Reference is made to the following:    Patent Literature 1: “Control of flow cytometer having vacuum fluidics”—U.S. Pat. No. 5,395,588 A    Patent Literature 2: “Method of aligning, compensating, and calibrating a flow cytometer for analysis of samples, and microbead standards kit therefor”—U.S. Pat. No. 5,093,234 A    Patent Literature 3: “Method for analysis of cellular components of a fluid”—U.S. Pat. No. 5,047,321 A    Patent Literature 4: “Apparatus for counting and/or measuring particles suspended in a fluid medium”—U.S. Pat. No. 4,056,324 A    Patent Literature 5: “Apparatus for measuring cytological properties”—U.S. Pat. No. 4,225,229 A    Patent Literature 6: “Orifice inside optical element”—U.S. Pat. No. 4,348,107 A    Patent Literature 7: “Particle Separator”—U.S. Pat. No. 3,380,584
Reference is also made to U.S. Pat. No. 4,395,676, filed Nov. 24, 1980, issued Jul. 26, 1983, entitled “Focused aperture module”; to U.S. Pat. No. 4,487,320, filed Nov. 3, 1980, issued Dec. 11, 1984, entitled “Method of and apparatus for detecting change in the breakoff point in a droplet generation system”; to U.S. Pat. No. 4,498,766, filed Mar. 25, 1982, issued Feb. 12, 1985, entitled “Light beam focal spot elongation in flow cytometry devices”; and to U.S. Pat. No. 3,657,537, filed Apr. 3, 1970, issued Apr. 18, 1972, entitled “Computerized slit-scan cyto-fluorometer for automated cell recognition”; to U.S. Pat. No. 8,159,670 to Vacca et al.; to U.S. Publication No. 2005046848A1; to U.S. Publication No. 2005057749; to U.S. Publication No. 20120270306; and to U.S. Publication No. 2012220022, each of which is incorporated herein by reference.
Reference is also made to the following:    1. Fulwyler M J. “Electronic Separation of Biological Cells by Volume”. Science 1965; 150: 910-911.    2. Fulwyler M J, Glascock, R B, Hiebert, R D, and Johnson N M. “Device which Separates Minute Particles According to Electronically Sensed Volume” 1969; Rev. Sci. Inst: 40: 42-48    3. Van Dilla M A, Mullaney P F, and Coulter J R. “Health Division Annual Report,” Los Alamos Scientific Laboratory (July 1966-June 1967).    4. Van Dilla M A, Trujillo T T, Mullaney P., and Coulter J R. “Cell Microfluorometry: A New Method for the Rapid Measurement of biological Cells Stained with Fluorescent Dyes.” Science 1969; 163: 1213-1214    5. Mullaney P F, Van Dilla M A, Coulter J R, and Dean P N. “A Light Scattering Photometer for Rapid Volume Determination.” Rev. Sci. Instr. 1969; 40: 1029-1032    6. Kay and Wheeless, “Experimental findings on Gynecologic cell orientation and dynamics for three flow nozzle geometries.” J. Histochem. Cytochem 1977, 25: 870
Further information about conventional flow cytometry can be found in Shapiro, H. M. Practical Flow Cytometry. John Wiley & Sons, Feb. 25, 2005.